Assay of free and complexed trypsinogen-2

ABSTRACT

The invention relates to an immunoassay for trypsinogen-2 wherein an amount of analyte in a sample is measured, said analyte being either trypsin-2 complexed with alpha-1-antitrypsin (trypsin-2-AAT) in serum, or trypsinogen-2 in urine. According to a preferred embodiment, the trypsin-2-AAT complex or free trypsinogen-2 are measured by non-competitive methods employing at least two different antibodies. The methods are useful for the diagnosis of patients with pancreatic disease, especially pancreatitis.

The present invention provides an immunoassay for the measurement oftrypsinogen-2 either as free trypsinogen-2 in urine or as its complexwith alpha-1-antitrypsin (trypsin-2-AAT) in serum. The invention relatesfurther to a method for differentiating between pancreatitis and otherpancreatic disease on one hand and non-pancreatic gastrointestinaldisease on the other hand by determining either free trypsinogen-2 inurine or trypsin -2-AAT in serum.

BACKGROUND OF THE INVENTION

The publications and other materials used herein to illuminate thebackground of the invention, and in particular, cases to provideadditional details respecting the practice, are incorporated byreference.

The trypsinogens are serine proteases secreted by exocrine cells of thepancreas (Travis J and Roberts R. Biochemistry 1969; 8: 2884-9; MalloryP and Travis J, Biochemistry 1973; 12: 2847-51). Two major types oftrypsinogen isoenzymes have been characterized, trypsinogen-1, alsocalled cationic trypsinogen, and trypsinogen-2 or anionic trypsinogen.The trypsinogen proenzymes are activated to trypsins in the intestine byenterokinase, which removes an activation peptide from the N-terminus ofthe trypsinogens. The trypsinogens show a high degree of sequencehomology, but they can be separated on the basis of charge differencesby using electrophoresis or ion exchange chromatography. The major formof trypsinogen in the pancreas and pancreatic juice is trypsinogen-1(Guy CO et al., Biochem Biophys Res Commun 1984; 125: 516-23). In serumof healthy subjects, trypsinogen-1 is also the major form, whereas inpatients with pancreatitis, trypsinogen-2 is more strongly elevated(Itkonen et al., J Lab Clin Med 1990; 115:712-8). Trypsinogens alsooccur in certain ovarian tumors, in which trypsinogen-2 is the majorform (Koivunen et al., Cancer Res 1990; 50: 2375-8). Trypsin-1 incomplex with alpha-1-antitrypsin, also called alpha-1-antiprotease, hasbeen found to occur in serum of patients with pancreatitis (Borgstrom Aand Ohlsson K, Scand J Clin Lab Invest 1984; 44: 381-6) butdetermination of this complex has not been found useful fordifferentiation between pancreatic and other gastrointestinal diseases(Borgstrom et al., Scand J Clin Lab Invest 1989; 49:757-62).

Trypsinogen-1 and -2 are closely related immunologically (Kimland etal., Clin Chim Acta 1989; 184: 31-46; Itkonen et al., 1990), but byusing monoclonal antibodies (Itkonen et al., 1990) or by absorbingpolyclonal antisera (Kimland et al., 1989) it is possible to obtainreagents enabling specific measurement of each form of trypsinogen.

When active trypsin reaches the blood stream, it is inactivated by themajor trypsin inhibitors alpha-2-macroglobulin and alpha-1-antitrypsin(AAT). AAT is a 58 kilodalton serine protease inhibitor synthesized inthe liver and is one of the main protease inhibitors in blood. Whereascomplexes between trypsin-1 and AAT are detectable in serum (Borgstromand Ohlsson, 1984) the complexes with alpha -2-macroglobulin are notmeasurable with antibody-based assays (Ohlsson K, Acta GastroenterolBelg 1988; 51: 3-12).

Acute pancreatitis is a severe and potentially lethal disease requiringrapid diagnosis. Determinations of amylase in serum or urine areroutinely used for this purpose, but these methods are hampered by lackof specificity, which limits their clinical usefulness. Therefore, theuse of other pancreatic markers, e.g. proteases and lipases have gainedinterest (Ogawa et al., Nippon Geka Gakkai Zassshi 1985; 86: 1241-4).Determination of trypsinogen-1 and -2 in serum (Itkonen et al., 1990)and trypsinogen-1 in urine (Lake-Bakaar et al., Lancet 1979; ii: 878-80)have been used for diagnosis of pancreatic disease.

In the present invention it has been shown that patients with pancreaticdisease have strongly elevated concentrations of trypsinogen-2 complexedwith alpha-1antitrypsin in serum and free trypsinogen-2 in urine.Accordingly, this discovery has proved to be very useful in thediagnosis of pancreatitis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents the characterization of the trypsin-2-alpha-1-antitrypsin complex in the calibrator by gel filtration. Theelution of trypsinogen-2 immunoreactivity is shown for comparison.Arrows indicate the elution positions for IgG and albumin.

FIG. 2 presents the dose-response curve for the immunofluorometric assayof trypsin -2-alpha-1-antitrypsin complex with the calibrator used.

FIG. 3A presents the concentration of trypsinogen-2 in serum samplesfrom 120 healthy controls, 11 patients with extrahepatic biliaryobstruction, 34 patients with acute abdominal disorders ofextrapancreatic origin and 29 patients with acute pancreatitis. Thehorizontal lines indicate the upper reference limits.

FIG. 3B presents the concentration of trypsin -2-alpha-1-antitrypsincomplex and serum amylase in the same patients as in FIG. 3A.

FIG. 4 presents a receiver operating characteristic (ROC) plots oftrypsin -2-AAT, trypsinogen-2 and amylase in sera from 29 patients withacute pancreatitis. Patients with acute abdominal disorders ofextrapancreatic origin were used as a control group. Values for somecut-off points are indicated. The figure demonstrates that trypsin-2-AAT has the best ability to discriminate between pancreatitis andnonpancreatic acute abdominal disorders.

FIG. 5 presents the concentration of trypsinogen-2 and amylase in urinesamples from 46 patients with acute abdominal disorders ofextrapancreatic origin and from 22 patients with acute pancreatitis. Thefigure demonstrates that trypsinogen-2 in urine has the best diagnosticaccuracy.

FIGS. 6A-6D show the concentrations of trypsinogen-2 and amylase inurine and serum samples from patients with mild pancreatitis (I) andsevere pancreatitis (II). Patients with acute abdominal disorders ofextrapancreatic origin served as controls (AC). HC=healthy controls. Thedashed horizontal line indicates the upper reference limit.

FIG.7A-7B illustrate ROC plots showing the accuracy of the various testsin differentiating between acute pancreatitis and extrapancreaticgastrointestinal disease (FIG. 7A) and between severe and mild acutepancreatitis (FIG. 7B).

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to an immunoassay for trypsinogen-2 wherein anamount of analyte in a sample is measured, said analyte being eithertrypsin-2 complexed with alpha-1-antitrypsin (trypsin -2-AAT) in serum,or trypsinogen-2 in urine.

Competitive as well as non-competitive assay methods can be employed.According to a preferred embodiment, the trypsin -2-AAT complex or freetrypsinogen-2 are measured by non-competitive methods employing at leasttwo different antibodies, where the capture antibody is bound to a solidphase.

Suitable labels are for example enzymes, radio-isotopes, fluorescent,phosforescent or luminescent markers, and colored particles that arevisibly detectable. The word "label" shall also be understood to cover abinding site able to bind to the markers mentioned.

The invention further concerns a substantially purified trypsin -2-AATcomplex and a substantially purified preparation of trypsinogen-2derived from urine.

Furthermore the invention concerns methods for differentiating betweenpancreatitis and other pancreatic diseases on one hand andnon-pancreatic disease on the other hand, either based on thedetermination of the concentration of trypsin -2-AAT in serum, or on theconcentration of trypsinogen-2 in urine.

The invention will be illuminated by the following non-restrictiveexamples.

Materials Used in the Examples Presented Below

1. Monoclonal antibodies

Monoclonal antibodies were produced in mice by standard procedures. Thespecificity of the antibodies for trypsinogen-2 was analyzed (Itkonen etal., 1990) using trypsinogen-1 and -2 separated by ion exchangechromatography as described in the reference (Koivunen et al., 1990).

2. Reagents and Buffers

The polyclonal antiserum against AAT was obtained from Dako (Glostrup,Denmark). The assay buffer in the immunofluorometric assay (IFXA) was 50mM Tris-HCl, pH 7.7, with 9 g/L NaCl (TBS) containing, per liter, 5 gbovine serum albumin, 0.15 g bovine globulin and 0.5 g NaN₃. The washsolution contained per liter, 9 g NaCl, 0.5 g NaN₃ and 0.2 g Tween20™(polyoxyethylenesorbitan monolaurate). Enhancement solution was fromWallac Biochemical laboratories (Turku, Finland).

3. Samples

Serum samples were obtained from 29 patients with acute pancreatitis, 11patients with extrahepatic biliary obstruction and 34 patients withacute abdominal disorders of extrapancreatic origin. The samples weredrawn within 24 hours of admission and before initiation of therapy. Thediagnosis of acute pancreatitis was based on clinical and laboratoryfindings (serum and urine amylase and C-reactive protein/CRP), Ransoncriteria (Ranson et al., Surg Gynecol Obstet 1974; 139: 69-80). Thediagnosis was confirmed by contrast computer tomography of the pancreas.The diagnosis of extrahepatic biliary obstruction and other acuteabdominal disorders of extrapancreatic origin were based on regularclinical methods (clinical, laboratory, ultrasonography and radiologicalexaminations or operative findings). Gastric and duodenal ulcers as wellas oesophagitis and gastritis were diagnosed by endoscopy. Serum samplesfrom 120 blood donors obtained from the Finnish Red Cross Blood Bankwere used to establish reference values. All samples were stored at--20° C. until assayed.

4. Gel Filtration

Fractionation of the trypsin -2-AAT calibrator was performed by gelfiltration using a 1x30 cm column of Superdex 200™ (a copolymer ofagarose and polyacrylamide) HR 10/30 (Pharmacia, Sweden) and TBS bufferfor elution. Flow rate was 30 mL/h and fractions of 0.5 ml volume werecollected. The tubes were prefilled with 50 μL assay buffer containingaprotinin (1.0 mg/L) in order to prevent proteolysis and nonspecificadsorption to the tubes. The elution volumes of IgG (150 kD) and albumin(69 kD) were used for a rough calibration of the column.

5. Detemination of Amylase

Amylase was measured by an enzymatic colorimetric test using anautomatic analyzer (Hitachi 705E) and reagents from Boehringer Mannheim.The reference values of the method are 70-300 U/L in serum and 60-2000U/L in urine.

EXAMPLE 1

Preparation of Trypsin -2-Alpha-1-Antitrypsin (Trypsin -2-AAT) for useas calibrator

A calibrator for the trypsin -2-AAT complex was prepared from pure humanAAT (Sigma Chemical Co., St. Louis, USA) and trypsin-2 purified asdescribed earlier (Itkonen et al., 1990) from urine of a patient withacute pancreatitis. Trypsinogen-2 was autoactivated at +37° C. for 2 hand then incubated for 16 h at +20° C. with a seven-fold molar excess ofAAT in TBS buffer. The incubation mixture was separated by gelfiltration and the content of trypsinogen-2 and trypsin -2-AAT in thefractions was estimated by trypsinogen-2 IFMA (FIG. 1). Incubation withAAT reduced the trypsinogen-2 component to 6% of the content in thecontrol incubated with aprotinin. The cross reaction in thetrypsinogen-2 IFXA with trypsin -2-AAT is 3.3% (FIG. 1). On the basis ofthis it was calculated that 94% of trypsin-2 had complexed with AAT.This preparation was used for calibration of the trypsin -2-AAT assay.

Calibrators were prepared by diluting the complex with assay buffer tocontain trypsin -2-AAT at concentrations of 0.1, 0.5, 1.0, 10, and 100μg/L.

EXAMPLE 2

Non-Competitive Immunofluorometric Assay of Trypsinogen-2 and Trypsin-2-AAT

Trypsinogen-2 was determined with a non-competitive time-resolvedimmunofluorometric assay using two monoclonal antibodies (Itkonen etal., 1990). The reference range for trypsinogen-2 in serum was 18-90μg/L (median 39 μg/L).

For assay of trypsin -2-AAT a monoclonal capture antibody to trypsin-2(14F10) (Itkonen et al, 1990) reacting both with free trypsinogen-2 andtrypsin -2-AAT complex was coated onto microtitration wells. Apolyclonal rabbit antibody to AAT (Dako, Denmark) labelled with aeuropium chelate (Hemmila et al, Anal Biochem 1984; 137: 335-43) wasused as detector antibody. Twenty-five μL of sample and 200 μL of assaybuffer were pipetted into the coated wells. After incubation for onehour the wells were emptied, washed twice with wash solution using anautomatic washer (DELFIA Platewash 1296-024, Wallac, Turku, Finland).Two-hundred ng of tracer antibody in 200 μL of assay buffer was added,and after further incubation for one hour the wells were emptied andwashed four times. 200 μL enhancement solution was added and after 5 minthe fluorescence was measured with a 1234 DELFIA fluorometer (Wallac,Turkut Finland). The dose-response curve for the trypsin -2-AAT IFMA isshown in FIG. 2.

The detection limit of the assay was 0.05 μg/L and the standard curvewas linear to 100 μg/L. The reference range determined on the basis ofthe 2.5 and 97.5 percentiles in sera from 120 blood donors was 2.3-12μg/L and the median value was 4.2 μg/L.

Trypsin -2-AAT and Trypsinogen-2 in Serum in the Diagnosis ofPancreatitis

The clinical accuracy of the serum assay was estimated by determiningthe concentrations of trypsin -2-AAT, trypsinogen-2 and amylase in serumsamples from patients with acute pancreatitis, patients withextrahepatic biliary obstruction and patients with acute abdominaldisorders of extrapancreatic origin. In patients with acute pancreatitisthe levels of trypsin -2-AAT and trypsinogen-2 were strongly elevated.The median concentration of trypsin -2-AAT was 59-fold that in healthycontrols. That of trypsinogen-2 was 19-fold and that of amylase 5.4-foldthat in healthy controls (Table 1).

                  TABLE 1                                                         ______________________________________                                        Median value and range of trypsin-2-AAT, trypsinogen-2 and                      amylase in serum from healthy controls and different                          patient groups (AAT abbreviation for alpha-1-antitrypsin).                          trypsin-2-AAT                                                                             trypsinogen-2                                                                              amylase                                        (μg/L) (μg/L) (U/L)                                                   Me-                 Me-            Me-                                          dian Range dian Range dian Range                                            ______________________________________                                        Healthy 4.2    2.1-14   39    11-233 180  60-300                                controls                                                                      Biliary 6.0 3.8-23 37 9.6-347 148 43-403                                      obstruction                                                                   Acute 7.6 2.4-36 27 7.6-145 178 78-711                                        abdominal                                                                     disorders                                                                     Acute 249 52-2170 750 75-4750 969 151-7020                                    pancreatitis                                                                ______________________________________                                    

There was no overlapping in trypsin -2-AAT values between the patientswith acute pancreatitis and those with extrapancreatic disease. Fortrypsinogen-2 overlapping was observed in 4 patients (14%) and foramylase in 14 patients (48%) (FIGS. 3A and 3B).

The ability of the various analytes to differentiate betweenpancreatitis and non-pancreatic disease was estimated byreceiver-operating characteristic (ROC) analysis. The area under the ROCcurve was 1.00 for trypsin -2-AAT , 0.996 for trypsinogen-2 and 0.929for amylase indicating that sensitivity and specificity was better fortrypsin -2-AAT than for the other determinations (FIG. 4).

EXAMPLE 3

a) Purification of Trypsinogen-2 from Urine

One liter of urine containing high concentrations of trypsinogen-2 wasfiltered to remove insoluble constituents and the pH of the filtrate wasadjusted to 7.4 with 1 mol/l NaOH. The filtrate was passed through animmunoaffinity chromatography column prepared by coupling 20 mg of themonoclonal antibody 14F10, which is specific for trypsinogen-2, tocyanogen bromide activated SEPHAROSE™ (a spherical agarose gel bead(from Pharmacia, Uppsala, Sweden). The characteristics of the antibodyhave been described (Itkonen et al. 1990). After washing of the columnwith 1 mol/l NaCl it was eluted with 0.1% trifluoroacetic acid (TFA).The eluate was further purified by reverse phase chromatography using0.1% TFA buffer and elution with an acetonitrile gradient. The contentof the fractions was monitored for protein on the basis of itsabsorbance at 280 nm. The fractions obtained were immediatelyneutralized with 1 mol/l Tris buffer. The content of trypsinogen-2 inthe fractions was determined by immunoassay. The fractions containingtrypsinogen-2 were concentrated. Trypsinogen-2 purified by this methodwas more than 95% pure as evidence by a single band in sodium dodecylsulfate electrophoresis.

b) Trypsinogen-2 in Urine in the Diagnosis of Pancreatitis

The clinical accuracy of the urine assay for trypsinogen-2 was estimatedby determining the concentrations in urine samples from 22 patients withacute pancreatitis and 46 patients with acute abdominal disorders ofextrapancreatic origin. Amylase in urine was used for comparison. Inpatients with acute pancreatitis the levels of trypsinogen-2 werestrongly elevated, the median concentrations being 4000-fold that inpatients with acute abdominal disorders. The corresponding differencefor amylase was only 4-fold (Table 2).

                  TABLE 2                                                         ______________________________________                                        Median value and range for trypsinogen-2 and amylase in                         urine samples of 46 patients with acute abdominal disorder                    of extrapancreatic origin and 22 patients with acute                          pancreatitis                                                                           Trypsinogen-2  Amylase                                               μg/L U/L                                                                          Median                                                                              Range      Median  Range                                       ______________________________________                                        Acute abdominal                                                                          2       0-525      878   140-13000                                   disorders                                                                     Acute pancreatitis 8100 790-195000 3900 170-69000                           ______________________________________                                    

There was no overlapping in trypsinogen-2 values between the patientswith acute pancreatitis and the control group with abdominal disordersof extrapancreatic origin. For amylase only 4 patients with pancreaticshad values higher than the highest ones observed in the control group(FIG. 5).

EXAMPLE 4

Urine Trypsinogen-2 as a Marker of Acute Pancreatitis Compared to SerumTrypsinogen-2 and Amylase in Serum or Urine

The clinical utility of determinations of urine trypsinogen-2 in theearly diagnosis and assessment of the severity of acute panceratitis(AP) was studied (FIG. 6A). As reference methods serum trypsinogen-2(FIG. 6B), urine amylase (FIG. 6C) and serum amylase (FIG. 6D) wereused. A total of 59 patients with a diagnosis of AP and 42 controls withacute upper abdominal disease of extrapancreatic origin (AC) werestudied at the Helsinki University Central Hospital. The patients withAP were classified according to their clinical outcome in two groups:mild AP (group I, 40 patients) and severe AP (group

II, 19 patients). Urine samples from patients without evidence of acuteabdominal disease were used as healthy controls (HC). Amylase andtrypsinogen were determined according to methods described before.

All cases of acute pancreatitis (AP) had elevated urine concentrationsof trypsinogen-2 at presentation (FIG. 6A) and a concentration below theupper reference value excluded this diagnosis. In contrast, 17 (28%) ofthe patients with pancreatitis had a normal level of amylase in urine(FIG. 6C). The lowest trypsinogen-2 value in acute pancreatitis (15μg/L) was 1.4-fold the upper reference limit in healthy controls. Themedian concentration of urine trypsinogen-2 in patients with AP was102-fold that of the upper reference limit (FIG. 6A). For comparison,the levels of urine amylase were only 2.0-fold (FIG. 6C) and that ofserum amylase 3.4-fold the upper reference limit (FIG. 6D).

Receiver operating characteristic analysis (ROC) was used to comparepatients with acute pancreatitis (AP) with controls having acuteabdominal extrapancreatic disorders (AC), AUC (area under the curve) was0.980 for trypsinogen-2 in urine and 0.842 for amylase in urine.Trypsinogen-2 in urine had as good accuracy as trypsinogen-2 in serum(AUC=0.998) and serum amylase (AUC=0.965), see FIG. 7A.

ROC-analysis showed that urine trypsinogen-2 had the highest accuracy ofthe markers studied in differentiating severe from mild AP (AUC=0.741,FIG. 7B) and it was slightly better than serum trypsinogen-2 in thisrespect. Amylase had poor ability to differentiate severe from mild APand the levels of urine amylase were actually lower in severe than inmild disease (FIG. 6C).

These results show that trypsinogen-2 in urine is a marker with highaccuracy for acute pancreatitis. All patients with acute pancreatitishave elevated values, and the number of false positive results fortrypsinogen-2 in patients with other gastrointestinal diseases is lowerthan for urine amylase. Furthermore the level of trypsinogen-2 reflectsthe severity of the disease, which the amylase level does not do. Thustrypsinogen-2 is clearly superior to other urine test in the diagnosisof acute pancreatitis and is equal to the serum test for trypsinogen-2.

It is obvious from the presented data that determination of trypsin-2-AAT in serum or trypsinogen-2 in urine provides better clinicalaccuracy than presently used methods such as trypsinogen-2 or amylase inserum or amylase in urine.

It will be appreciated that the methods of the present invention can beincorporated in the form of a variety of embodiments, only a few ofwhich are disclosed herein. It will be apparent for the expert skilledin the field that other embodiments exist and do not depart from thespirit of the invention. Thus, the described embodiments areillustrative and should not be construed as restrictive.

We claim:
 1. A method for determining whether a human patient has acutepancreatitis, wherein said method comprises the steps of:(a) (i)measuring a concentration of trypsin-2 complexed with α₁ -antitrypsin(trypsin-2-AAT) in a serum sample from said patient by contacting saidserum sample from said patient with a capture antibody specific for saidtrypsin-2-AAT to form an immune complex correlative of the concentrationof said trypsin-2-AAT in said serum sample from said patient, and(ii)comparing said concentration of said trypsin-2-AAT in said serum samplefrom said patient with a range of concentrations of said trypsin-2-AATin serum samples from healthy humans measured with said captureantibody, or (b) (i) measuring a concentration of free trypsinogen-2 ina urine sample from said patient by contacting said urine sample fromsaid patient with a capture antibody specific for said trypsinogen-2 toform an immune complex correlative of the concentration of said freetrypsinogen-2 in said urine sample from said patient, and(ii) comparingsaid concentration of said free trypsinogen-2 in said urine sample fromsaid patient with a range of concentrations of said free trypsinogen-2in urine samples from healthy humans measured with said captureantibody, wherein when (a) said concentration of said trypsin-2-AAT insaid serum sample from said patient or (b) said concentration of saidfree trypsinogen-2 in said urine sample from said patient is greaterthan said respective range of concentrations, then said patient hasacute pancreatitis.
 2. The method according to claim 1 wherein saidcontacting is performed in the presence of (a) a labeled trypsin-2-AATor (b) a labeled free trypsinogen-2 which competes with said (a)trypsin-2-AAT or (b) free trypsinogen-2 for binding to said captureantibody.
 3. The method according to claim 2 wherein said labeledtrypsin-2-AAT or said labeled trypsinogen-2 comprises a label selectedfrom the group consisting of an enzyme, a radioisotope, a fluorophore, aphosphore, a luminophore, and a colored particle.
 4. The methodaccording to claim 2 wherein said labeled trypsin-2-AAT or said labeledtrypsinogen-2 is indirectly labeled with an enzyme, a radioisotope, afluorophore, a phosphore, a luminophore or a colored particle.
 5. Themethod according to claim 1 wherein said contacting is performed in thepresence of (a) a labeled detector antibody specific for saidtrypsin-2-AAT or (b) a labeled detector antibody specific for said freetrypsinogen-2 wherein said capture antibody and said labeled detectorantibody are each specific for a different second site on saidtrypsin-2-AAT or said free trypsinogen-2.
 6. The method according toclaim 5 wherein said capture antibody is bound to a solid phase prior tosaid contacting.
 7. The method according to claim 6 wherein saidtrypsin-2-AAT is measured, said serum sample from said patient issequentially contacted first with said capture antibody and then withsaid labeled detector antibody, and further wherein AAT not complexedwith said trypsin-2 is removed before the step of contacting with saidlabeled detector antibody.
 8. The method according to claim 7 whereinsaid capture antibody is specific for the trypsin-2 moiety of saidtrypsin-2-AAT.
 9. The method according to claim 7 wherein said labeleddetector antibody is specific for AAT when said AAT is complexed withsaid trypsin-2.
 10. The method according to claim 5 wherein said captureantibody is specific for the trypsin-2 moiety of said trypsin-2-AAT. 11.The method according to claim 5 wherein said trypsin-2-AAT is measured,said serum sample from said patient is sequentially contacted first withsaid capture antibody and then with said labeled detector antibody, andfurther wherein AAT not complexed with said trypsin-2 is removed beforethe step of contacting with said labeled detector antibody.
 12. Themethod according to claim 11 wherein said capture antibody is specificfor the trypsin-2 moiety of said trypsin-2-AAT.
 13. The method accordingto claim 12 wherein said labeled detector antibody is specific for AATwhen said AAT is complexed with said trypsin-2.
 14. The method accordingto claim 11 wherein said labeled detector antibody is specific for AATwhen said AAT is complexed with said trypsin-2.
 15. The method accordingto claim 1 wherein said capture antibody is bound to a solid phase priorto said contacting.
 16. The method according to claim 1 wherein saidcapture antibody is specific for the trypsin-2 moiety of saidtrypsin-2-AAT.